Alzheimer's disease (AD) causes dementia in many elderly individuals and in individuals with Down's syndrome who survive to age 50. AD is characterized by several distinct pathological features that are visible on histological examination: large numbers of amyloid plaques surrounded by neurons containing neurofibrillary tangles and neuronal cell loss.
One hypothesis regarding the pathogenesis of the disease is that deposition of amyloid .beta. polypeptide (.beta.AP), which is the major macromolecular component of amyloid plaques, is the causative agent of the characteristic AD pathological changes leading to formation of neurofibrillary tangles, neuronal cell loss, vascular damage, and, ultimately, dementia (Hardy and Higgins (1992) Science 256: 184). Amyloid precursor protein (APP) is encoded by a single gene in humans. RNA transcripts of the APP gene are alternatively spliced to encode several APP protein isoforms; the predominant APP isoform in brain lacks a serine protease inhibitor domain that is present in other tissues. .beta.AP is a proteolytic cleavage product arising from the carboxy region of various APP isoforms, including the predominant APP isoform in the brain (Kitaguchi et al. (1988) Nature 331: 530; Ponte et al., ibid., p.525; R. E. Tanzi, ibid., p.528; Kang and Muller-Hill (1990) Biochem. Biophys. Res. Commun. 166: 1192; Yoshioka et al. (1991) Biochem. Biophys. Res. Commun. 178: 1141; Johnson et al. (1990) Science 248: 854; Neve et al. (1990) Neuron 5: 329). According to this hypothesis, amyloidogenic .beta.AP might be generated by one or more alternate proteolytic cleavage pathways. The accumulation of extracellular .beta.AP results in insoluble amyloid deposits and may be neurotoxic, leading to neuronal death and neurofibrillary tangle formation.
The serine protease inhibitor (i.e., serine antiproteinase) alpha 1-antichymotrypsin (.alpha.1-ACT), which binds to chymotrypsin-like enzymes in a covalent manner, has been shown recently to be both a normal constituent of brain and an integral component of the amyloid neuritic plaques that form in Down's syndrome and AD (Abraham and Potter (1989) Ann. Med. 21: 77; Abraham et al. (1988) Cell 52: 487; Pasternack et al. (1989) Am. J. Pathol. 135: 827; Matsubara et al. (1990) Ann. Neurol. 28: 561; Koo et al. (1991) Neurobiology of Aging 12: 495). Since the neuritic plaques which characterize AD comprise .beta.AP, which is presumably a proteolytic cleavage product of one or more APP isoforms (such as the major brain isoform which lacks a serine protease inhibitor domain), the presence of .alpha.1-ACT in amyloid plaques suggests that it may play a role in either normal or pathological proteolytic processing of APP isoforms.
Serine antiproteinases, also termed serpins, encompass a supergene family of proteinase inhibitors that regulate many of the serine proteases involved in inflammation and homeostasis. Alpha 1-antichymotrypsin belongs to the serpin supergene family that includes .alpha.1-antitrypsin (.alpha.1-AT), antithrombin III (ATIII), ovalbumin, angiotensinogen, human leuserpin 2 (hLS2), protein C inhibitor (plasminogen activator inhibitor III), rat kallikrein binding protein (RKBP), mouse contrapsin, and several other related proteins (e.g., murine Spi-2 genes).
The human serine protease inhibitor genes .alpha.1-AT and .alpha.1-ACT are acute-phase proteins which are induced in response to inflammation. These inhibitors function to limit the activity of specific serine proteases in vivo; .alpha.1-AT acts as an inhibitor of neutrophil elastase to protect the elastin fibers of the lung. The physiologic role of .alpha.1-ACT is not clearly defined but it likely functions in modulating the enzymatic activity of one or more specific serine protease(s). Human .alpha.1-ACT is a plasma protease inhibitor that specifically inactivates serine proteases of the chymotrypsin class, including cathepsin G and other proteases found in neutrophils, basophils, and tissue mast cells. Human .alpha.1-ACT is produced in liver, and it possesses the unusual feature of being a primary acute-phase protein whose plasma concentration may rise several-fold within 24 hours after tissue damage. These facts suggest that the primary role of .alpha.1-ACT is in regulating chymotrypsin-like enzymes, particularly those released during inflammatory episodes.
Human .alpha.1-ACT has been cloned, sequenced, and expressed in E. coli (Rubin et al. (1990) J. Biol. Chem. 26.5: 1199; Kelsey et al. (1988) J. Med. Genet. 25: 361). The human .alpha.1-ACT gene is approximately 12 kilobases (kb) in length and contains five exons and four introns (Bao et al. (1987) Biochemistry 26: 7755; Ragg and Preibisch (1988) J. Biol. Chem. 263: 12129). The human .alpha.1-ACT gene maps to the same region, q31-32.3 of chromosome 14, that the human .alpha.1-AT gene maps to (Rabin et al. (1986) Somat. Cell Mol. Genet. 12: 209) and the two genes are located within about 220 kb of each other (Sefton et al. (1990) Genomics 7: 382).
The homologous organization of genes of the serpin supergene family (Chai et al. (1991) J. Biol. Chem. 266: 16029; Meijers and Chung (1991) 266: 15028), the high degree of sequence homology between their protein sequences (Chandra et al. (1983) Biochemistry 22: 5055; Chao et al. (1990) J. Biol. Chem. 265: 16394; Suzuki et al. (1990) J. Biochem. 108: 344), and clustering of serpin genes at particular chromosal locations (Rabin et al. (1986) op.cit.; Sefton et al. (1990) op.cit.) suggests that these genes arose by recent gene duplication events (Bao et al. (1987) op.cit.).
Human .alpha.1-ACT are glycoproteins encoded by the single copy .alpha.1-ACT gene, which are typically about 68,000 daltons and comprise about 25-35% carbohydrate. .alpha.1-ACT is found in whole blood, serum, plasma, cerebrospinal fluid (CSF), and brain tissue. The predominant plasma form of .alpha.1-ACT is presumed to be synthesized in the liver. Proteolytically modified .alpha.1-ACT has been crystallized, and Asn70 and Asn104 have been identified as putative glycosylation sites (Baumann et al. (1991) J. Mol. Biol. 218: 595).
Two predominant isoforms of .alpha.1-ACT have been identified in plasma and correspond to a microheterogeneity at the amino-terminus of .alpha.1-ACT that can be identified by the presence of two well-separated components of about 68 kD on immunoelectrophoresis (Travis et al. (1978) Biochemistry 17: 5647). Most investigators report that the major isoform has an amino-terminal arginine (Travis (1978) op.cit.; Laine and Hayem (1981) Biochim. Biophys. Acta 6.68.: 429). However, one report, Morii and Travis (1983) Biochem. Biophys. Res. Commun. 111: 438, found that over 90 percent of .alpha.1-ACT has an amino-terminal aspartic acid and less than 10 percent has an amino-terminal arginine, with a fifteen amino acid peptide fragment being cleaved off the amino-terminus of the major isoform (N-terminal aspartic acid) to yield the minor form (N-terminal arginine). A more recent report, Lindmark et al. (1989) Biochim. Biophys. Acta 997: 90, indicates that one .alpha.1-ACT ACT isoform has the amino-terminal sequence (His-Pro-Asn-Ser-Pro-(SEQ. ID. NO:1)) and the other isoform is truncated by two residues and has the amino-terminal sequence (Asn-Ser-Pro). .alpha.1-ACT purified from normal human serum has also been separated by affinity chromatography on a Concanavalin-A-Sepharose column into three microheterogenous forms that differ by their N-linked carbohydrate structures (Laine et al. (1991) Eur. J. Biochem. 197: 209). Hence, there appear to be several discrete forms of .alpha.1-ACT in human plasma.
Immunocytochemical staining has identified one or more proteins in amyloid deposits in AD neuritic plaques that are reactive with antibodies to .alpha.1-ACT. In situ hybridization studies using probes corresponding to an .alpha.1-ACT cDNA isolated from a liver cDNA library have identified astrocytes as a source of mRNA that hybridizes to .alpha.1-ACT cDNA probes, with astrocytes surrounding neuritic plaque cores having intense labeling by the probe (Pasternack (1989) op.cit.; Koo et al. (1991) op.cit.). The levels of mRNA that hybridize to a liver .alpha.1-ACT cDNA in brain gray matter has been shown to be elevated in AD brain versus control brain (Abraham (1988) op.cit.). It is not known, however, if the .alpha.1-ACT mRNA or protein(s) synthesized in brain are structurally equivalent to or distinct from one or more of the forms of .alpha.1-ACT synthesized by the liver and present in the plasma.
The diagnosis of Alzheimer's disease requires a detailed clinical evaluation, and diagnosis generally cannot be made until significant symptoms of dementia and memory loss are clinically apparent. Unfortunately, no practicable in vitro biochemical diagnostic test is presently available for diagnosing AD in its early stages and for identifying AD candidates. Koo et al. (1991) op.cit. report data indicating that increased expression of RNA that hybridizes to .alpha.1-ACT DNA probes in the brain increases with age and is associated with astrocytosis. Expression of mRNA that hybridizes to .alpha.1-ACT DNA probes is apparently associated with astrocytosis and/or astrogliosis, which characterize many different neuropathological conditions (e.g., Alzheimer's disease and certain CNS neoplasms). Matsubara et al. (1990) op.cit. were unable to demonstrate that measurement of .alpha.1-ACT in serum is a sufficiently selective and sensitive method for identifying AD patients, and alternatively have suggested that while measuring serum levels of .alpha.1-ACT may eventually be useful as a biochemical diagnostic marker of AD, there is presently no useful biochemical marker available. Moreover, even the equivocal results of Matsubara et al. have not yet proven to be reproducible.
Therefore, since .alpha.1-ACT mRNA levels in brain tissue are increased in AD and other neurodegenerative disorders, there exists a need in the art for methods and compositions for identifying and quantitating .alpha.1-ACT species that are produced in brain tissue and for distinguishing the brain .alpha.1-ACT signal from the background liver-produced .alpha.1-ACT present in the serum. These methods and compositions could be applied to develop diagnostic methods and kits for detecting the presence of or predisposition to a neurological disease, such as a neurodegenerative disease (e.g., Alzheimer's disease), or for use as cell type-specific markers for cells derived from the central nervous system (e.g., astroglial cells), such as in metastatic brain tumors.